LISTERIA HEMOLYSIN AND ESCAPE FROM A VACUOLE

性状（改编自申请人摘要）：单核细胞增生李斯特菌 是一种典型的兼性细胞内病原体，主要感染 孕妇和免疫功能低下的人。 的主要决定因素 L.单核细胞增多症的发病机制是一种分泌的孔形成蛋白， 作为溶血素O（LLO）。 LLO主要是造成 由吞噬作用产生的寄主液泡。 产气荚膜梭菌溶素O（PFO）是一种 相关的孔形成蛋白，其参与了 被细胞外病原体感染。 当用L. PFO介导单核细胞增多症，PFO介导从吞噬泡逃逸，但有毒 到细胞。 通常，LLO在感染期间持续表达，但 与PFO相反，它在胞质溶胶中被蛋白水解降解， 与MHC I类分子联合呈递在细胞表面。 它 假设最适pH和胞质处理是必不可少的 LLO编码的决定因素，使其与PFO区分开来。 目前建议的重点是确定准确的结构和 LLO的机械特征，使其区别于其他成员， 巯基活化溶细胞素家族，促进其液泡内活性， 并在细胞质中引导其命运。 在目标I中，负责 对于LLO的酸性pH最佳值和在宿主胞质溶胶中的加工， 鉴定 这将通过域和子域交换来完成 LLO和PFO之间的突变，以及修饰的电荷-丙氨酸扫描诱变。 LLO/PFO嵌合体将从E.大肠杆菌，并表征 生物化学 接下来，将嵌合体引入L。单核 并在感染的组织培养模型中表征。 在Aim II中，宿主胞质溶胶中LLO加工的途径将被完全阻断。 评估。 LLO将通过L的代谢标记来识别。单核 在感染的宿主细胞内，随后进行免疫沉淀。 将通过脉冲追踪确定前体/产物关系 实验 将使用特异性抑制剂来评估 蛋白酶体和其他蛋白酶的降解。 LLO的作用 将对磷酸化进行生物化学和遗传学评价。 在目标III中，L的确切性质。单核细胞增生吞噬体 特征在于pH、穿孔时间和穿孔的标记物。 内体/溶酶体成熟途径。 最适pH和L. 单核细胞增生性磷脂酶将通过使用突变和嵌合 菌株 在目标IV中，研究者将评估使用E.杆菌 K12表达LLO和重组蛋白作为一种新的系统， 外源蛋白进入哺乳动物胞质溶胶用于抗原呈递。

DESCRIPTION (Adapted from the applicant's abstract): Listeria monocytogenes is a model facultative intracellular pathogen which primarily infects pregnant women and immunocompromised individuals. A primary determinant of L. monocytogenes pathogenesis is a secreted pore-forming protein referred to as listeriolysin O (LLO). LLO is largely responsible for rupture of the host vacuole which results from phagocytosis. Perfringolysin O (PFO) is a related pore-forming protein which is involved in the pathogenesis of infections by an extracellular pathogen. When expressed by L. monocytogenes, PFO mediates escape from the phagocytic vacuole, but is toxic to the cell. Normally, LLO is continually expressed during infection, but in contrast to PFO, it is proteolytically degraded in the cytosol and presented on the cell surface in association with MHC class I molecules. It is hypothesized that pH optimum and cytosolic processing are essential LLO-encoded determinants which distinguish it from PFO. The focus of the current proposal is to define the precise structural and mechanistic features of LLO which differentiate it from other members of the family of thiol-activated cytolysins, facilitate its intravacuolar activity, and direct its fate in the cytosol. In Aim I, protein sequences responsible for LLO's acidic pH optimum and processing in the host cytosol will be identified. This will be accomplished by domain and sub-domain swapping between LLO and PFO, and modified charged-to-alanine scanning mutagenesis. The LLO/PFO chimeras will be purified from E. coli and characterized biochemically. Next, the chimeras will be introduced into L. monocytogenes and characterized in tissue culture models of infection., In Aim II, the pathway of LLO processing in the host cytosol will be fully evaluated. LLO will be identified by metabolic labeling of L. monocytogenes within infected host cells, followed by immunoprecipitation. Precursor/product relationships will be determined by pulse-chase experiments. Specific inhibitors will be used to evaluate the role of the proteosome and other proteases in degradation. The role of LLO phosphorylation will be evaluated biochemically and genetically. In Aim III, the precise nature of the L. monocytogenes phagosome will be characterized with regard to pH, time of perforation, and markers of the endosome/lysosome pathway of maturation. The role of pH optimum and the L. monocytogenes phospholipases will be evaluated by using mutant and chimeric strains. In Aim IV, the investigators will evaluate the feasibility of using E. coli K12 expressing LLO and a recombinant protein as a novel system to introduce foreign proteins into the mammalian cytosol for antigen presentation.